Arabidopsis antisense deoxyhypusine synthase molecule and method of inhibiting deoxyhypusine synthase expression in plants

ABSTRACT

Regulation of expression of programmed cell death, including senescence, in plants is achieved by integration of a gene or gene fragment encoding senescence-induced deoxyhypusine synthase, senescence-induced eIF-5A or both into the plant genome in antisense orientation. Plant genes encoding senescence-induced deoxyhypusine synthase and senescence-induced eIF-5A are identified and the nucleotide sequences of each, alone and in combination are used to modify senescence in transgenic plants.

This application is a divisional application of Ser. No. 09/597,771,filed on Jun. 19, 2000, now U.S. Pat. No. 6,538,182, which is acontinuation-in-part application of Ser. No. 09/348,675, filed on Jul.6, 1999, now abandoned.

FIELD OF THE INVENTION

The present invention relates to polynucleotides which encode plantpolypeptides that exhibit senescence-induced expression. The inventionalso relates to transgenic plants containing the polynucleotides inantisense orientation and methods for controlling programmed cell death,including senescence, in plants. More particularly, the presentinvention relates to a senescence induced plant deoxyhypusine synthasegene and a senescence-induced eIF-5A gene whose expressions are inducedby the onset of programmed cell death, including senescence, and the useof the deoxyhypusine synthase gene and eIF-5A gene, alone or incombination, to control programmed cell death and senescence in plants.

DESCRIPTION OF THE PRIOR ART

Senescence is the terminal phase of biological development in the lifeof a plant. It presages death and occurs at various levels of biologicalorganization including the whole plant, organs, flowers and fruit,tissues and individual cells.

The onset of senescence can be induced by different factors bothinternal and external. Senescence is a complex, highly regulateddevelopmental stage in the life of a plant or plant tissue, such asfruit, flowers and leaves. Senescence results in the coordinatedbreakdown of cell membranes and macromolecules and the subsequentmobilization of metabolites to other parts of the plant.

In addition to the programmed senescence which takes place during normalplant development, death of cells and tissues and ensuing remobilizationof metabolites occurs as a coordinated response to external,environmental factors. External factors that induce premature initiationof senescence, which is also referred to as necrosis or apoptosis,include environmental stresses such as temperature, drought, poor lightor nutrient supply, as well as pathogen attack. Plant tissues exposed toenvironmental stress also produce ethylene, commonly known as stressethylene (Buchanan-Wollaston, V., 1997, J. Exp. Botany, 48:181-199;Wright, M., 1974, Plant, 120:63-69). Ethylene is known to causesenescence in some plants.

Senescence is not a passive process, but, rather, is an activelyregulated process that involves coordinated expression of specificgenes. During senescence, the levels of total RNA decrease and theexpression of many genes is switched off (Bate et al., 1991, J. Exper.Botany, 42, 801-11; Hensel et al., 1993, The Plant Cell, 5, 553-64).However, there is increasing evidence that the senescence processdepends on de novo transcription of nuclear genes. For example,senescence is blocked by inhibitors of mRNA and protein synthesis andenucleation. Molecular studies using mRNA from senescing leaves andgreen leaves for in vitro translation experiments show a changed patternof leaf protein products in senescing leaves (Thomas et al, 1992, J.Plant Physiol., 139, 403-12). With the use of differential screening andsubtractive hybridization techniques, many cDNA clones representingsenescence-induced genes have been identified from a range of differentplants, including both monocots and dicots, such as Arabidopsis, maize,cucumber, asparagus, tomato, rice and potato. Identification of genesthat are expressed specifically during senescence is hard evidence ofthe requirement for de novo transcription for senescence to proceed.

The events that take place during senescence appear to be highlycoordinated to allow maximum use of the cellular components beforenecrosis and death occur. Complex interactions involving the perceptionof specific signals and the induction of cascades of gene expressionmust occur to regulate this process. Expression of genes encodingsenescence related proteins is probably regulated via common activatorproteins that are, in turn, activated directly or indirectly by hormonalsignals. Little is known about the mechanisms involved in the initialsignaling or subsequent co-ordination of the process.

Coordinated gene expression requires factors involved in transcriptionand translation, including initiation factors. Translation initiationfactor genes have been isolated and characterized in a variety oforganisms, including plants. Eukaryotic translation initiation factor 5A(eIF-5A) is an essential protein factor approximately 17 KDa in size,which is involved in the initiation of eukaryotic cellular proteinsynthesis. It is characterized by the presence of hypusine[N-(4-amino-2-hydroxybutyl)lysine], a unique modified amino acid, knownto be present only in eIF-5A. Hypusine is formed post-translationallyvia the transfer and hydroxylation of the butylamino group from thepolyamine, spermidine, to the side chain amino group of a specificlysine residue in eIF-5A. Activation of eIF-5A involves transfer of thebutylamine residue of spermidine to the lysine of eIF-5A, forminghypusine and activating eIF-5A. In eukaryotes, deoxyhypusine synthase(DHS) mediates the post-translational synthesis of hypusine in eIF-5A. Acorresponding DHS gene has not been identified in plants, however, it isknown that plant eIF-5A contains hypusine. The hypusine modification hasbeen shown to be essential for eIF-5A activity in vitro using amethionyl-puromycin assay.

Hypusine is uniquely present in eIF-5A and is found in all eukaryotes,some archaebacteria (which appear to be related to eukaryota), but notin eubacteria. Moreover, the amino acid sequence of eIF-5A is highlyconserved, especially in the region surrounding the hypusine residue,suggesting that eIF-5A and its activating protein, deoxyhypusinesynthase, execute fundamentally important steps in eukaryotic cellphysiology (Joe et al., JBC, 270:22386-22392, 1995). eIF-5A has beencloned from human, alfalfa, slime mold, Neurospora crassa, tobacco andyeast. It was originally identified as a general translation initiationfactor based on its isolation from ribosomes of rabbit reticulocytelysates and its in vitro activity in stimulating methionine-puromycinsynthesis. However, more recent data indicate that eIF-5A is not atranslation initiation factor for global protein synthesis, but ratherserves to facilitate the translation of specific subsets of mRNApopulations. For example, there is strong evidence from experiments withanimal cells and yeast that one or more isoforms of eIF-5A play anessential role in mediating the translation of a subset of mRNAsinvolved in cell proliferation. There are two isoforms in yeast, and ifboth genes are silenced the cells are unable to divide (Park et al.,Biol. Signals, 6:115-123, 1997). Similarly, silencing the expression ofyeast deoxyhypusine synthase, which activates eIF-5A, blocks celldivision. Indeed, inhibitors of deoxyhypusine synthase have beendeveloped that are likely to have importance in the therapy ofhyperproliferative conditions (Wolff, et al., JBC, 272:15865-15871,1997). Other studies have indicated that another isoform of eIF-5A isessential for Rev function in HIV-1 replication or Rex function in HTLVV replication (Park, et al., Biol. Signals, 6:115-123, 1997). There arealso at least two expressed eIF-5A genes in tobacco. Gene-specificprobes indicate that although they are both expressed in all tissuesexamined, each gene has a distinctive expression pattern, presumablyregulating the translation of specific transcripts (Chamot, et al., Nuc.Acids Res., 20:625-669, 1992).

Deoxyhypusine synthase has been purified from rat testis, HeLa cells,Neurospora crassa and yeast. The amino acid sequence of deoxyhypusinesynthase is highly conserved, and the enzymes from different speciesshare similar physical and catalytic properties and displaycross-species reactivities with heterologous eIF-5A precursors (Park, etal., 6 Biol. Signals, 6:115-123, 1997).

Plant polyamines have been implicated in a wide variety of physiologicaleffects including floral induction, embryogenesis, pathogen resistance,cell growth, differentiation and division (Evans et al., 1989, Annu.Rev. Plant Physiol. Plant Mol. Biol., 40, 235-269; and Galston, et al.,1990, Plant Physiol., 94, 406-10). It has been suggested that eIF-5A isthe intermediary through which polyamines exert their effects (Chamot etal., 1992, Nuc. Acids Res., 20(4), 665-69).

Two genes encoding isoforms of eIF-5A from Nicotiana have beenidentified (NeIF-5A1 and NeIF-5A2) (Chamot et al., 1992, Nuc. AcidsRes., 20(4), 665-69). The genes were shown to be very similar. However,they display differential patterns of expression. One gene appears to beconstitutively expressed at the mRNA level, while the expression patternof the other correlates with the presence or absence of photosyntheticactivity. Based on gene structure and genomic Southern mapping it hasbeen suggested that there is a multigene family of NeIF-5A genes intobacco. It is likely that there is an eIF-5A isoform that regulatestranslation of a subset of senescence/necrosis specific mRNAtranscripts.

Presently, there is no widely applicable method for controlling theonset of programmed cell death (including senescence) caused by eitherinternal or external, e.g., environmental stress, factors. It is,therefore, of interest to develop senescence modulating technologiesthat are applicable to all types of plants and that are effective at theearliest stages in the cascade of events leading to senescence.

SUMMARY OF THE INVENTION

This invention is based on the discovery and cloning of a full lengthcDNA clone encoding a tomato senescence-induced deoxyhypusine synthase(DHS), as well as full length senescence-induced DHS cDNA clones fromArabidopsis leaf and carnation petal. The nucleotide sequences andcorresponding amino acid sequences are disclosed herein.

The invention is also based, in part, on the discovery and cloning offull length cDNA clones encoding a senescence-induced eIF-5A gene fromtomato, Arabidopsis and carnation. The nucleotide sequence andcorresponding amino acid sequence of each of the eIF-5A cDNA clones aredisclosed herein.

The present invention provides a method for genetic modification ofplants to control the onset of senescence, either age-related senescenceor environmental stress-induced senescence. The senescence-induced DHSnucleotide sequences of the invention, fragments thereof, orcombinations of such fragments, are introduced into a plant cell inreverse orientation to inhibit expression of the endogenoussenescence-induced DHS gene, thereby reducing the level of endogenoussenescence-induced DHS protein, and reducing and/or preventingactivation of eIF-5A and ensuing expression of the genes that mediatesenescence.

In another aspect of the invention, the senescence-induced eIF-5Anucleotide sequences of the invention, fragments thereof, orcombinations of such fragments, are introduced into a plant cell inreverse orientation to inhibit expression of the endogenoussenescence-induced eIF-5A gene, and thereby reduce the level ofendogenous senescence-induced eIF-5A protein, and reduce and/or preventensuing expression of the genes that mediate senescence. Alternatively,both DHS sequences and eIF-5A sequences can be used together to reducethe levels of endogenous DHS and eIF-5A proteins

In yet another aspect, the present invention is directed to a method forgenetic modification of plants to control the onset of senescence,either age-related senescence or environmental stress-induced senescencevia the introduction into a plant cell of a combination ofsenescence-induced eIF-5A nucleotide sequences of the invention andsenescence-induced DHS nucleotide sequences of the invention in reverseorientation to inhibit expression of the endogenous senescence-inducedeIF-5A gene and senescence-induced DHS gene, thereby reducing the levelof endogenous senescence-induced DHS protein, and reducing and/orpreventing activation of eIF-5A and ensuing expression of the genes thatmediate senescence.

Using the methods of the invention, transgenic plants are generated andmonitored for growth, development and either natural orprematurely-induced senescence. Plants or detached parts of plants(e.g., cuttings, flowers, vegetables, fruits, seeds or leaves)exhibiting prolonged life or shelf life, (e.g., extended life offlowers, reduced fruit or vegetable spoilage, enhanced biomass,increased seed yield, reduced seed aging and/or reduced yellowing ofleaves) due to reduction in the level of senescence-induced DHS,senescence-induced eIF-5A or both are selected as desired productshaving improved properties including reduced leaf yellowing, reducedpetal abscission, reduced fruit and vegetable spoilage during shippingand storage. These superior plants are propagated. Similarly, plantsexhibiting increased resistance to environmental stress, e.g., decreasedsusceptibility to low temperature (chilling), drought, infection, etc.,and/ or increased resistance to pathogens, are selected as superiorproducts.

In one aspect, the present invention is directed to an isolated DNAmolecule encoding senescence-induced DHS, wherein the DNA moleculehybridizes with SEQ ID NO:1, or a functional derivative of the isolatedDNA molecule which hybridizes with SEQ ID NO:1. In one embodiment ofthis aspect of the invention, the isolated DNA molecule has thenucleotide sequence of SEQ ID NO:1, i.e., 100% complementarity (sequenceidentity) to SEQ ID NO:1.

The present invention also is directed to an isolated DNA moleculeencoding senescence-induced DHS, wherein the DNA molecule hybridizeswith SEQ ID NO:9, or a functional derivative of the isolated DNAmolecule which hybridizes with SEQ ID NO:9. In one embodiment of thisaspect of the invention, the isolated DNA molecule has the nucleotidesequence of SEQ ID NO:9, i.e., 100% complementarity (sequence identity)to SEQ ID NO:9.

The present invention also is directed to an isolated DNA moleculeencoding senescence-induced eIF-5A, wherein the DNA molecule hybridizeswith SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15 or a functional derivativeof the isolated DNA molecule which hybridizes with SEQ ID NO:11, SEQ IDNO:13, SEQ ID NO:15. In one embodiment of this aspect of the invention,the isolated DNA molecule has the nucleotide sequence of SEQ ID NO:11,SEQ ID NO:13, or SEQ ID NO:15, i.e., 100% complementarity (sequenceidentity) to SEQ ID NO:11, SEQ ID NO:13 or SEQ ID NO:15.

In another embodiment of the invention, there is provided an isolatedprotein encoded by a DNA molecule as described herein above, or afunctional derivative thereof. A preferred protein has the amino acidsequence of SEQ ID NO:2, or is a functional derivative thereof. Anotherpreferred protein has the amino acid sequence of SEQ ID NO:10, or is afunctional derivative thereof. Other preferred proteins of the inventionhave the amino acid sequence of SEQ ID NO:12, SEQ ID NO:14 or SEQ ID NO:16.

Also provided herein is an antisense oligonucleotide or polynucleotideencoding an RNA molecule which is complementary to a correspondingportion of an RNA transcript of a DNA molecule described herein above,wherein the oligonucleotide or polynucleotide hybridizes with the RNAtranscript such that expression of endogenous senescence-induced DHS isaltered. In another embodiment of this aspect of the invention, theantisense oligonucleotide or polynucleotide is an RNA molecule thathybridizes to a corresponding portion of an RNA transcript of a DNAmolecule described herein above, such that expression of endogenoussenescence-induced eIF-5A is altered. The antisense oligonucleotide orpolynucleotide can be full length or preferably has about six to about100 nucleotides.

The antisense oligonucleotide or polynucleotide may be substantiallycomplementary to a corresponding portion of one strand of a DNA moleculeencoding senescence-induced DHS, wherein the DNA molecule encodingsenescence-induced DHS hybridizes with SEQ ID NO:1, SEQ ID NO: 5, SEQ IDNO: 9, or with a combination thereof, or is substantially complementaryto at least a corresponding portion of an RNA sequence encoded by theDNA molecule encoding senescence-induced DHS. In one embodiment of theinvention, the antisense oligonucleotide or polynucleotide issubstantially complementary to a corresponding portion of one strand ofthe nucleotide sequence SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:9 or with acombination thereof, or the RNA transcript transcribed from SEQ ID NO:1,SEQ ID NO:5, SEQ ID NO:9 or with a combination thereof. In anotherembodiment, the antisense oligonucleotide is substantially complementaryto a corresponding portion of the 5′ non-coding portion or 3′ portion ofone strand of a DNA molecule encoding senescence-induced DHS, whereinthe DNA molecule hybridizes with SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:9or with a combination thereof.

Alternatively, the antisense oligonucleotide or polynucleotide may besubstantially complementary to a corresponding portion of one strand ofa DNA molecule encoding senescence-induced eIF-5A, wherein the DNAmolecule encoding senescence-induced eIF-5A hybridizes with SEQ IDNO:11, SEQ ID NO:13, SEQ ID NO:15, or any combination thereof, or issubstantially complementary to at least a corresponding portion of anRNA sequence transcribed from SEQ ID NO:11, SEQ ID NO:13 or SEQ IDNO:15. In one embodiment of the invention, the antisense oligonucleotideor polynucleotide is substantially complementary to a correspondingportion of one strand of the nucleotide sequence SEQ ID NO:11, SEQ IDNO:13, SEQ ID NO:15 or a combination thereof, or the RNA transcriptencoded is substantially complementary to a corresponding portion of anRNA sequence encoded by a DNA molecule encoding senescence-inducedeIF-5A. In another embodiment, the antisense oligonucleotide issubstantially complementary to a corresponding portion of the 5′non-coding region or 3′ region of one strand of a DNA molecule encodingsenescence-induced eIF-5A, wherein the DNA molecule hybridizes with SEQID NO:11, SEQ ID NO:13, SEQ ID NO:15 or a combination thereof.

The invention is further directed to a vector for transformation ofplant cells, comprising

(a) an antisense oligo- or polynucleotide substantially complementary to(1) a corresponding portion of one strand of a DNA molecule encodingsenescence-induced DHS, wherein the DNA molecule encodingsenescence-induced DHS hybridizes with SEQ ID NO:1, SEQ ID NO:5 or SEQID NO:9, or (2) a corresponding portion of an RNA sequence encoded bythe DNA molecule encoding senescence-induced DHS; and

(b) regulatory sequences operatively linked to the antisense oligo- orpolynucleotide such that the antisense oligo- or polynucleotide isexpressed in a plant cell into which it is transformed.

The invention is further directed to a vector for transformation ofplant cells, comprising

(a) an antisense oligo- or polynucleotide substantially complementary to(1) a corresponding portion of one strand of a DNA molecule encodingsenescence-induced eIF-5A, wherein the DNA molecule encodingsenescence-induced eIF-5A hybridizes with SEQ ID NO:11, SEQ ID NO:13,SEQ ID NO:15 or (2) a corresponding portion of an RNA sequence encodedby the DNA molecule encoding senescence-induced eIF-5A; and

(b) regulatory sequences operatively linked to the antisense oligo- orpolynucleotide such that the antisense oligo- or polynucleotide isexpressed in a plant cell into which it is transformed.

The regulatory sequences include a promoter functional in thetransformed plant cell, which promoter may be inducible or constitutive.Optionally, the regulatory sequences include a polyadenylation signal.

The invention also provides a plant cell transformed with a vector orcombination of vectors as described above, a plantlet or mature plantgenerated from such a cell, or a plant part of such a plantlet or plant.

The present invention is further directed to a method of producing aplant having a reduced level of senescence-induced DHS,senescence-induced eIF-5A or both compared to an unmodified plant,comprising:

(1) transforming a plant with a vector or combination of vectors asdescribed above;

(2) allowing the plant to grow to at least a plantlet stage;

(3) assaying the transformed plant or plantlet for alteredsenescence-induced DHS activity and/or eIF-5A activity and/or alteredsenescence and/or altered environmental stress-induced senescence and/orpathogen-induced senescence and/or ethylene-induced senescence; and

(4) selecting and growing a plant having altered senescence-induced DHSactivity and/or reduced eIF-5A and/or altered senescence and/or alteredenvironmental stress-induced senescence and/or altered pathogen-inducedsenescence and/or ethylene-induced senescence compared to anon-transformed plant.

Plants produced as above, or progeny, hybrids, clones or plant partspreferably exhibit reduced senescence-induced DHS expression, reducedsenescence-induced eIF-5A activity, or both and delayed senescenceand/or delayed stress-induced senescence and/or pathogen-inducedsenescence and/or ethylene-induced senescence.

This invention is further directed to a method of inhibiting expressionof endogenous senescence-induced DHS in a plant cell, said methodcomprising:

(1) integrating into the genome of a plant a vector comprising

-   -   (A) an antisense oligo- or polynucleotide complementary to (I)        at least a portion of one strand of a DNA molecule encoding        endogenous senescence-induced DHS, wherein the DNA molecule        encoding the endogenous senescence-induced DHS hybridizes with        SEQ ID NO:1, SEQ ID NO:5 and/or SEQ ID NO.9, or (ii) at least a        portion of an RNA sequence encoded by the endogenous        senescence-induced DHS gene; and    -   (B) regulatory sequences operatively linked to the antisense        oligo- or polynucleotide such that the antisense oligo- or        polynucleotide is expressed; and

(2) growing said plant, whereby said antisense oligo- or polynucleotideis transcribed and the transcript binds to said endogenous RNA wherebyexpression of said senescence-induced DHS gene is inhibited.

This invention is further directed to a method of inhibiting expressionof endogenous senescence-induced eIF-5A in a plant cell, said methodcomprising:

(1) integrating into the genome of a plant a vector comprising

-   -   (A) an antisense oligo- or polynucleotide complementary to (I) a        corresponding portion of one strand of a DNA molecule encoding        endogenous senescence-induced eIF-5A, wherein the DNA molecule        encoding the endogenous senescence-induced eIF-5A hybridizes        with SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:17 or a combination        thereof, or (ii) at least a portion of an RNA sequence encoded        by the endogenous senescence-induced eIF-5A gene; and    -   (B) regulatory sequences operatively linked to the antisense        oligo- or polynucleotide such that the antisense oligo- or        polynucleotide is expressed; and

(2) growing said plant, whereby said antisense oligo- or polynucleotideis transcribed and the transcript binds to said endogenous RNA wherebyexpression of said senescence-induced eIF-5A gene is inhibited.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the nucleotide sequence of the senescence-induced tomatoleaf DHS cDNA sequence (SEQ ID NO:1) and the derived amino acid sequence(SEQ ID NO. 2) obtained from a tomato leaf cDNA library.

FIG. 2A depicts the nucleotide sequence of an Arabidopsis DHS geneobtained by aligning the tomato DHS sequence with unidentified genomicsequences in the Arabidopsis gene bank (SEQ ID NO:5). The gaps betweenamino acid sequences are predicted introns. FIG. 2B depicts the derivedArabidopsis DHS amino acid sequence (SEQ ID NO:6). FIG. 2C depicts thenucleotide sequence of a 600 base pair senescence-induced ArabidopsisDHS cDNA obtained by PCR. FIG. 2D depicts the derived amino acidsequence of the senescence-induced Arabidopsis DHS cDNA fragment.

FIG. 3 is an alignment of the derived full length tomato leafsenescence-induced DHS amino acid sequence (SEQ ID NO. 2) and thederived full length Arabidopsis senescence-induced DHS amino acidsequence with sequences of DHS proteins of human, yeast, fungi, andArchaeobacteria. Identical amino acids among three or four of thesequences are boxed.

FIG. 4 is a restriction map of the tomato DHS cDNA.

FIG. 5 is a Southern blot of genomic DNA isolated from tomato leaves andprobed with ³²P-dCTP-labeled full length tomato senescence-induced DHScDNA.

FIG. 6 is a Northern blot of RNA isolated from tomato flowers atdifferent stages of development. FIG. 6A is the ethidium bromide stainedgel of total RNA. Each lane contains 10 μg RNA. FIG. 6B is anautoradiograph of the Northern blot probed with ³²P-dCTP-labeled fulllength tomato senescence-induced DHS cDNA.

FIG. 7 is a Northern blot of RNA isolated from tomato fruit at variousstages of ripening that was probed with ³²P-dCTP-labelled full lengthtomato senescence-induced DHS cDNA. Each lane contains 10 μg RNA.

FIG. 8 is a Northern blot of RNA isolated from tomato leaves that hadbeen drought-stressed by treatment with 2 M sorbitol for six hours. Eachlane contains 10 μg RNA. The blot was probed with ³²P-dCTP-labelled fulllength tomato senescence-induced DHS cDNA.

FIG. 9 is a Northern blot of RNA isolated from tomato leaves that hadbeen exposed to chilling temperature. FIG. 9A is the ethidium bromidestained gel of total RNA. Each lane contained 10 μg RNA. FIG. 9B is anautoradiograph of the Northern blot probed with ³²P-dCTP-labelled fulllength tomato senescence-induced DHS cDNA. FIG. 9C shows correspondingleakage data measured as conductivity of leaf diffusates.

FIG. 10 is the carnation DHS full-length (1384 base pairs) cDNA clonenucleotide sequence (SEQ ID NO: 9), not including the PolyA tail and 5′end non-coding region. The derived amino acid sequence is shown belowthe nucleotide sequence (373 amino acids). (SEQ ID NO:10)

FIG. 11 is a Northern blot of total RNA from senescing Arabidopsisleaves probed with ³²P-dCTP-labelled full-length Arabidopsissenescence-induced DHS cDNA. The autoradiograph is at the top, theethidium stained gel below.

FIG. 12 is a Northern blot of total RNA isolated from petals ofcarnation flowers at various stages. The blot was probed with³²P-dCTP-labelled full-length carnation senescence-induced DHS cDNA. Theautoradiograph is at the top, the ethidium stained gel below.

FIG. 13 is the nucleotide (top) (SEQ ID NO:11) and derived amino acid(bottom) (SEQ ID NO:12) sequence of the tomato fruit senescence-inducedeIF-5A gene.

FIG. 14 is the nucleotide (top) (SEQ ID NO:13) and derived amino acid(bottom) (SEQ ID NO:14) sequence of the carnation senescence-inducedeIF-5A gene.

FIG. 15 is the nucleotide (top) (SEQ ID NO:15) and derived amino acid(bottom) (SEQ ID NO:16) sequence of the Arabidopsis senescence-inducedeIF-5A gene.

FIG. 16 is a Northern blot of total RNA isolated from leaves ofArabidopsis plants at various developmental stages. The blot was probedwith ³²P-dCTP-labelled full-length Arabidopsis senescence-induced DHScDNA and full-length senescence-induced eIF-5A. The autoradiograph is atthe top, the ethidium stained gel below.

FIG. 17 is a Northern blot of total RNA isolated from tomato fruit atbreaker (BK), red-firm (RF) and red-soft (RS) stages of development. Theblot was probed with ³²P-dCTP-labelled full-length senescence-inducedDHS cDNA and full-length senescence-induced eIF-5A. DHS and eIF-5A areup-regulated in parallel in red-soft fruit coincident with fruitripening. The autoradiograph is at the top, the ethidium stained gelbelow.

FIG. 18 is a Northern blot of total RNA isolated from leaves of tomatothat were treated with sorbitol to induce drought stress. C is control;S is sorbitol treated. The blot was probed with ³²P-dCTP-labelledfull-length senescence-induced DHS cDNA and full-lengthsenescence-induced eIF-5A. Both eIF-5A and DHS are up-regulated inresponse to drought stress. The autoradiograph is at the top, theethidium stained gel below.

FIG. 19 is a Northern blot of total RNA isolated from flower buds andopen senescing flowers of tomato plants. The blot was probed with³²P-dCTP-labelled full-length senescence-induced DHS cDNA andfull-length senescence-induced eIF-5A. Both eIF-5A and DHS areup-regulated in open/senescing flowers. The autoradiograph is at thetop, the ethidium stained gel below.

FIG. 20 is a Northern blot of total RNA isolated from chill-injuredtomato leaves. The blot was probed with ³²P-dCTP-labelled full-lengthsenescence-induced DHS cDNA and full-length senescence-induced eIF-5A.Both eIF-5A and DHS are up-regulated with the development of chillinginjury during rewarming The autoradiograph is at the top, the ethidiumstained gel below.

FIG. 21 is a photograph of 3.1 week old Arabidopsis wild-type (left) andtransgenic plants expressing the 3′-end of the senescence DHS gene(sequence shown in FIG. 36) in antisense orientation showing increasedleaf size in the transgenic plants.

FIG. 22 is a photograph of 4.6 week old Arabidopsis wild-type (left) andtransgenic plants expressing the 3′-end of the senescence DHS gene(sequence shown in FIG. 36) in antisense orientation showing increasedleaf size in the transgenic plants.

FIG. 23 is a photograph of 5.6 week old Arabidopsis wild-type (left) andtransgenic plants expressing the 3′-end of the senescence DHS gene(sequence shown in FIG. 36) in antisense orientation showing increasedleaf size in the transgenic plants.

FIG. 24 is a photograph of 6.1 week old Arabidopsis wild-type (left) andtransgenic plants expressing the 3′-end of the senescence DHS gene(sequence shown in FIG. 36) in antisense orientation showing increasedsize of transgenic plants.

FIG. 25 is a graph showing the increase in seed yield from three T₁transgenic Arabidopsis plant lines expressing the senescence-induced DHSgene in antisense orientation. Seed yield is expressed as volume ofseed. SE for n=30 is shown for wild-type plants.

FIG. 26 is a photograph of transgenic tomato plants expressing the3′-end of the senescence DHS gene (sequence shown in FIG. 36) inantisense orientation (left) and wild-type plants (right) showingincreased leaf size and increased plant size in the transgenic plants.The photograph was taken 18 days after transfer of the plantlets tosoil.

FIG. 27 is a photograph of transgenic tomato plants expressing the3′-end of the senescence DHS gene (sequence shown in FIG. 36) inantisense orientation (left) and wild-type plants (right) showingincreased leaf size and increased plant size in the transgenic plants.The photograph was taken 32 days after transfer of the plantlets tosoil.

FIGS. 28 through 35 are photographs of tomato fruit from wild-type (toppanels) and transgenic plants expressing the full-length senescence DHSgene in antisense orientation (bottom panels). Fruit were harvested atthe breaker stage of development and ripened in a growth chamber. Daysafter harvest are indicated in the upper left corner of each panel.

FIG. 36 is the nucleotide (top) (SEQ ID NO:30) and derived amino acid(bottom) sequence of the 3′-end of the Arabidopsis senescence-inducedDHS gene used in antisense orientation to to transform plants.

FIG. 37 is the nucleotide (top) (SEQ ID NO:31) and derived amino acid(bottom) sequence of the 3′-end of the tomato senescence-induced DHSgene used in antisense orientation to transform plants.

FIG. 38 is the nucleotide (top) (SEQ ID NO:26) and derived amino acid(bottom) sequence of a 600 base pair Arabidopsis senescence-induced DHSprobe used to isolate the full-length Arabidopsis gene.

FIG. 39 is the nucleotide (top) (SEQ ID NO:27) and derived amino acid(bottom) sequence of the 483 base pair carnation senescence-induced DHSprobe used to isolate the full-length carnation gene.

DETAILED DESCRIPTION OF THE INVENTION

Methods and compositions are provided for altering the expression ofsenescence-induced DHS gene(s), senescence-induced eIF-5A gene(s) orboth in plant cells. Alteration of expression of senescence-induced DHSand senescence-induced eIF-5A, either alone or in combination, in plantsresults in delayed onset of senescence and improved resistance toenvironmental stress and pathogens, thus extending the plant shelf-lifeand/or growth period.

A full length cDNA sequence encoding a tomato DHS gene exhibitingsenescence-induced expression has been isolated by reverse transcriptasemediated polymerase chain reaction (RT-PCR) using RNA isolated fromchill-injured tomato leaves as a template and using the RT-PCR productto screen a chill-injured, sorbitol-treated tomato leaf cDNA library.Polynucleotide probes corresponding to selected regions of the isolatedtomato leaf cDNA sequence as well as the full length tomato leaf cDNAwere used to determine the presence of mRNA encoding the DHS gene inenvironmentally stressed (chilled) tomato leaves, (dehydrated)sorbitol-treated tomato leaves, ripening tomato fruit and senescingtomato blossoms.

Primers designed from an Arabidopsis DHS genomic clone were used togenerate a polymerase chain reaction (PCR) product using a senescingArabidopsis leaf cDNA library as template. The Arabidopsis nucleotidesequence has 73% nucleotide sequence identity and 81% amino acidsequence identity with the corresponding sequence of thesenescence-induced tomato DHS.

The senescence-induced tomato DHS gene of the present invention wasisolated by using RT-PCR. The upstream primer used to isolate the tomatoDHS gene is a 24 nucleotide primer: 5′ AG TCT AGA AGG TGC TCG TCC TGA T3′ (SEQ ID NO. 3); the downstream primer contains 34 nucleotides: 5′ GACT GCA GTC GAC ATC GAT (T)₁₅ 3′ (SEQ ID NO. 4). Using 100 pmol of thedownstream primer, a first strand of cDNA was isolated using standardRT-PCR. The first strand was then used as template in a RT-PCR, usingboth the upstream and downstream primers. Separation of the RT-PCRproducts on an agarose gel revealed the presence of three distinct bandsranging in size from 1.5 kb to 600 bp. The three fragments weresubcloned into the plasmid vector, pBluescript™ (Stratagene CloningSystems, LaJolla, Calif.) using XbaI and SalI cloning sites present inthe upstream and downstream primers, respectively, and sequenced. Thesequences of the fragments were compared and aligned with sequencespresent in the GeneBank data base. The results showed the 1.5 kb and 1kb fragments to be tomato DHS sequence. The 600 bp fragment also alignedwith human, yeast and Neurospora DHS sequences.

The 600 bp RT-PCR fragment was used to screen a tomato (cv. Match F1hybrid) cDNA library made from RNA obtained from tomato leaves that hadbeen treated with 2 M sorbitol for six hours to induce dehydration. ThecDNA library was constructed using a λZap™ (Stratagene Cloning Systems,LaJolla, Calif.) cDNA library kit. Three identical positive full-lengthcDNA clones corresponding to the senescence-induced DHS gene wereobtained and sequenced. The nucleotide sequence of thesenescence-induced DHS cDNA clone is shown in SEQ ID NO:1. The cDNAclone encodes a 381 amino acid polypeptide (SEQ ID NO: 2) having acalculated molecular mass of 42.1 KDa.

Based on the expression pattern of the gene in developing and stressedtomato flowers, fruit and leaves, it is involved in senescence.

The tomato DHS cDNA sequence was aligned with unidentified genomicsequences in the Arabidopsis thaliana genome bank The results showedalignment with an unidentified Arabidopsis genomic sequence (AB107060).The alignment information was used to identify an open reading frame inthe Arabidopsis sequence and generate predicted amino acid sequencetherefrom. The resulting nucleotide and amino acid sequences of thealigned Arabidopsis DHS gene are designated as SEQ ID NO. 5 (FIG. 2A)and SEQ ID NO. 6, respectively.

Two primers based on short regions of the identified Arabidopsis DHSsequence were generated: primer 1, 5′ GGTGGTGTTGAGGAAGATC 3′ (SEQ ID NO.7); and primer 2, 5′ GGTGCACGCCCTGATGAAGC 3′ (SEQ ID NO. 8). AnArabidopsis senescing leaf cDNA library was used as template for the twoprimers in a standard PCR. A 600 bp PCR product was isolated andsequenced and shown to have an identical sequence as that of thecorresponding fragment of the genomic DHS sequence.

The full-length senescence-induced tomato DHS cDNA clone was also usedto isolate full-length senescence-induced Arabidopsis and carnation DHScDNA clones. The Arabidopsis and carnation DHS cDNA clones were isolatedby screening a senescing Arabidopsis leaf cDNA library and asenescencing carnation petal cDNA library, respectively, using thefull-length tomato DHS cDNA clone as probe. cDNA clones obtained fromthe cDNA libraries were then sequenced. The nucleotide sequence of theArabidopsis full-length cDNA clone isolated in this manner has the samesequence as the coding region of the Arabidopsis genomic sequenceidentified as encoding Arabidopsis DHS by alignment with the tomato cDNAsequence. (FIG. 2A, SEQ ID NO: 5). The nucleotide sequence of thefull-length carnation petal senescence-induced DHS clone and derivedamino acid sequence are shown in FIG. 10 (SEQ ID NO:9 and SEQ ID NO:10,respectively).

Thus, the cDNA sequences of the invention, encoding DHS from tomato,carnation and Arabidopsis can be used as probe in a similar manner toisolate DHS genes from other plants, which can then be used to altersenescence in transgenic plants.

The senescence-induced DHS gene appears to be a member of a DHS genefamily. Genomic Southern blot analysis of tomato leaf DNA was carriedout using genomic DNA extracted from a hybrid plant. The DNA was cutwith various restriction enzymes that recognize a single site within thecoding region of the DHS gene or which do not recognize any sites withinthe open reading frame of the DHS gene. A restriction map for tomato DHSis shown in FIG. 4.

Restriction enzyme digested tomato leaf genomic DNA was probed with³²P-dCTP-labeled full length tomato DHS cDNA. Hybridization under highstringency conditions revealed hybridization of the full length cDNAprobe to two to three restriction fragments for each restriction enzymedigested DNA sample. Of particular note, when tomato leaf genomic DNAwas digested with XbaI and EcoRI, which have restriction sites withinthe open reading frame of DHS (FIG. 4), more than two restrictionfragments were detectable in the Southern blot (FIG. 5). Genomic DNAfrom cv Match F1, a hybrid variety, and the homozygous line, UCT5,yielded the same pattern of restriction fragments. These results suggestthat there are two or more isoforms of the DHS gene in tomato plants. Asshown in FIG. 3, the DHS gene is highly conserved across species and soit would be expected that there is a significant amount of conservationbetween isoforms within any species.

Northern blots of tomato flower total RNA probed with the full lengthtomato cDNA show that the expression of the senescence-induced DHS geneis significantly induced in tomato blossoms, but expression is barelydetectable in the buds (FIG. 6). Northern blot analysis of DHSexpression during various developmental stages of tomato fruitdemonstrate that the DHS gene is expressed at low levels in breaker andpink fruit, whereas DHS expression in red (ripe) tomato fruit issignificantly enhanced (FIG. 7).

Northern blot analyses also demonstrate that the senescence-induced DHSgene is induced by environmental stress conditions, e.g., dehydration(FIG. 8) and chilling (FIG. 9). Tomato leaves that had been treated with2 M sorbitol to induce dehydration demonstrate induction of DHSexpression in the dehydrated leaves compared to non-treated leaves (FIG.8). Plants that have been exposed to chilling temperatures and returnedto ambient temperature show induced expression of the senescence-inducedDHS gene coincident with the development of chilling injury symptoms(e.g., leakiness) (FIG. 9). The overall pattern of gene expression intomato plants and various plant tissues, e.g., leaves, fruit andflowers, demonstrates that the DHS gene of the invention is involved inthe initiation of senescence in these plants and plant tissues.

Similar results in terms of induction of DHS gene expression areobserved with the onset of leaf senescence in Arabidopsis and petalsenescence in carnation. Northern blot analyses of Arabidopsis leaftotal RNA isolated from plants of various ages show that the expressionof the senescence-induced DHS gene is not evident in young(five-week-old plants), but begins to appear at about six weeks.Expression of the DHS gene is significantly induced by seven weeks.Northern blot analysis indicates that the Arabidopsis DHS gene issignificantly enhanced as the plant ages. (FIG. 11).

Northern blot analyses also demonstrate that the DHS gene is similarlyregulated in flowering plants, such as the carnation. (FIG. 12) Northernblot analyses of total RNA isolated from petals of carnation flowers ofvarious ages show that the expression of carnation DHS is significantlyinduced in petals from flowers that have symptoms of age-inducedsenescence such as petal inrolling, which is the first morphologicalmanifestation of senescence, but expression is much lower in tight-budflowers. Petals from carnation flowers that are just beginning to openhave significantly more DHS expression than flowers in the tight-budstage, and petals from flowers that are fully open also show enhancedexpression of DHS.

Thus, it is expected that by substantially repressing or altering theexpression of the senescence-induced DHS gene in plant tissues,deterioration and spoilage can be delayed, increasing the shelf-life ofperishable fruits, flowers, and vegetables, and plants and their tissuescan be rendered more stress-tolerant and pathogen resistant. This can beachieved by producing transgenic plants in which the DHS cDNA or anoligonucleotide fragment thereof is expressed in the antisenseconfiguration in fruits, flowers, leaves and vegetables, preferablyusing a constitutive promoter such as the CaMV 35S promoter, or using atissue-specific or senescence/stress-inducible promoter.

Another gene, eIF-5A, which is involved in the induction of senescencerelated morphological changes in plants has also been isolated andsequenced herein and like the DHS, it can be used to alter senescenceand senescence-related processes in plants, preferably, by introductionin antisense orientation into plants. A full-length senescence-inducedeIF-5A cDNA clone was isolated from,each of ripening tomato fruit,senescing Arabidopsis leaf and senescing carnation flower cDNAlibraries. The nucleotide and derived amino acid sequences of each ofthe full length clones is shown in FIG. 13 (tomato senescence-inducedeIF-5A), 14 (carnation senescence-induced eIF-5A) and 15 (Arabidopsissenescence-induced eIF-5A). The nucleotide sequence of each of thesecDNA clones is also shown as SEQ ID NO: 11 (tomato) (FIG. 13), SEQ IDNO:13 (carnation) (FIG. 14) and SEQ ID NO:15 (Arabidopsis) (FIG. 15).The derived amino acid sequence of each of the genes is shown as SEQ IDNO:12 (FIG. 13), SEQ ID NO:14 (FIG. 14) and SEQ ID NO:16 (FIG. 15),respectively.

As is the case with the DHS gene sequences described herein, the eIF-5Asequence of the present invention can be used to isolate eIF-5A genesfrom other plants. The isolated eIF-5A sequences can be used to altersenescence and senescence-related processes in plants. Isolation ofeIF-5A sequences from plants can be achieved using art known methods,based on sequences similarities of at least about 70% across species.

Parallel induction of eIF-5A and DHS occurs in plants during senescence.Northern blot analyses demonstrate that eIF-5A is upregulated inparallel with DHS at the onset of both natural and stress-inducedsenescence. (FIGS. 16 through 20) For example, Northern blot analyses oftotal RNA isolated from leaves of Arabidopsis plants at various agesdemonstrate that from the time leaf senescence is evident in the plantthe expression of eIF-5A is induced and expression is significantlyenhanced as senescence progresses. In fruit bearing plants, such astomato, eIF-5A and DHS are upregulated in parallel in red-soft fruitcoincident with the onset of fruit softening and spoilage. (FIG. 17)

Northern blot analysis also demonstrates that eIF-5A and DHS areupregulated in parallel in plants in response to environmantal stress,such as drought (FIG. 18) and chilling injury (FIG. 20). Similarly, inflowering plants, eIF-5A and DHS are upregulated in parallel in openflowers and expression of both genes continues to be enhanced throughthe later stages of flowering.

The cloned senescence-induced DHS gene, fragment(s) thereof, or clonedsenescence-induced eIF-5A gene or fragment(s) thereof, or combinationsof eIF-5A and DHS sequences, when introduced in reverse orientation(antisense) under control of a constitutive promoter, such as the figwart mosaic virus 35S promoter, cauliflower mosaic virus promoterCaMV35S, double 35S promoter or MAS promoter, can be used to geneticallymodify plants and alter senescence in the modified plants. Selectedantisense sequences from other plants which share sufficient sequenceidentity with the tomato, Arabidopsis or carnation senescence-inducedDHS genes or senscence-induced eIF-5A genes can be used to achievesimilar genetic modification. One result of the genetic modification isa reduction in the amount of endogenous translatable senescence-inducedDHS-encoding mRNA, eIF-5A-encoding mRNA or both. Consequently, theamount of senescence-induced DHS and/or senescence-induced eIF-5Aproduced in the plant cells is reduced, thereby reducing the amount ofactivated eIF-5A, which in turn reduces translation of senescenceinduced proteins, including senescence-induced lipase,senescence-induced proteases and senescence-induced nucleases.Senescence is thus inhibited or delayed, since de novo protein synthesisis required for the onset of senescence.

For example, Arabidopsis plants transformed with vectors that expresseither the full-length or 3′-region of the Arabidopsissenescence-induced DHS gene (SEQ ID NO:26) (FIG. 38) in antisenseorientation, under regulation of a double 35S promoter exhibit increasedbiomass, e.g., larger leaf size and overall larger plant growththroughout all stages of growth, and delayed leaf senescence incomparison to control plants as shown in FIGS. 21 through 24.

The effect of reduced expression of the senescence-induced DHS genebrought about by expressing either the full-length or 3′ coding regionof the Arabidopsis senescence-induced DHS gene in antisense orientationin transgenic Arabidopsis plants is also seen as an increase in seedyield in the transformed plants. Arabidopsis plant lines expressing theantisense 3′ non-coding region of the Arabidopsis senescence-induced DHSgene produce up to six times more seed than wild type plants. (FIG. 25)

Similar results are obtained with tomato plants transformed with the 3′end of the tomato senescence-induced DHS gene (SEQ ID NO:27) inantisense orientation and under regulation of a double 35S promoter.Plants transformed with the 3′ end of the gene in antisense orientationshow increased leaf size and increased plant size in comparison tocontrol (non-transformed) tomato plants. (FIGS. 26 and 27)

Tomato plants transformed with the full length tomato senescence-inducedDHS in antisense orientation produce fruit that exhibits delayedsoftening and spoilage in comparison to wild type plants. (FIGS. 28through 35). Thus, the methods and sequences of the present inventioncan be used to delay fruit softening and spoilage, as well as toincrease plant biomass and seed yield and in general, delay senesence inplants.

The isolated nucleotide sequences of this invention can be used toisolate substantially complementary DHS and'or eIF-5A nucleotidesequence from other plants or organisms. These sequences can, in turn,be used to transform plants and thereby alter senescence of thetransformed plants in the same manner as shown with the use of theisolated nucleotide sequences shown herein.

The genetic modifications obtained with transformation of plants withDHS, eIF-5A, fragments thereof or combinations thereof can effect apermanent change in levels of senescence-induced DHS, eIF-5A or both inthe plant and be propagated in offspring plants by selfing or otherreproductive schemes. The genetically altered plant is used to produce anew variety or line of plants wherein the alteration is stablytransmitted from generation to generation. The present inventionprovides for the first time the appropriate DNA sequences which may beused to achieve a stable genetic modification of senescence in a widerange of different plants.

For the identification and isolation of the senescence-induced DHS geneand eIF-5A gene, in general, preparation of plasmid DNA, restrictionenzyme digestion, agarose gel electrophoresis of DNA, polyacrylamide gelelectrophoresis of protein, PCR, RT-PCR, Southern blots, Northern blots,DNA ligation and bacterial transformation were carried out usingconventional methods well-known in the art. See, for example, Sambrook,J. et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold SpringHarbor Press, Cold Spring Harbor, N.Y., 1989. Techniques of nucleic acidhybridization are disclosed by Sambrook (Supra).

As used herein, the term “plant” refers to either a whole plant, a plantpart, a plant cell or a group of plant cells. The type of plant whichcan be used in the methods of the invention is not limited and includes,for example, ethylene-sensitive and ethylene-insensitive plants; fruitbearing plants such as apricots, apples, oranges, bananas, grapefruit,pears, tomatoes, strawberries, avocados, etc.; vegetables such ascarrots, peas, lettuce, cabbage, turnips, potatoes, broccoli, asparagus,etc.; flowers such as carnations, roses, mums, etc.; agronomic cropplants and forest species such as corn, rice, soybean, alfalfa and thelike; and in general, any plant that can take up and express the DNAmolecules of the present invention. It may include plants of a varietyof ploidy levels, including haploid, diploid, tetraploid and polyploid.The plant may be either a monocotyledon or dicotyledon.

A transgenic plant is defined herein as a plant which is geneticallymodified in some way, including but not limited to a plant which hasincorporated heterologous or homologous senescence-induced DHS DNA ormodified DNA or some portion of heterologous senescence-induced DHS DNAor homologous senescence-induced DHS DNA into its genome. Alternativelya transgenic plant of the invention may have incorporated heterologousor homologous senescence-induced eIF-5A DNA or modified DNA or someportion of heterologous senescence-induced eIF-5A DNA or homologoussenescence-induced eIF-5A DNA into its genome. Transgenic plants of theinvention may have incorporated heterologous or homologoussenescence-induced DHS and eIF-5A DNA or modified DNA or some portion ofheterologous senescence-induced DHS and eIF-5A DNA or homologoussenescence-induced DHS DNA or a combination of heterologous andhomologous DHS and eIF-5A sequences into its genome. The altered geneticmaterial may encode a protein, comprise a regulatory or controlsequence, or may be or include an antisense sequence or encode anantisense RNA which is antisense to the endogenous senescence-inducedDHS or eIF-5A DNA or mRNA sequence or portion thereof of the plant. A“transgene” or “transgenic sequence” is defined as a foreign gene orpartial sequence which has been incorporated into a transgenic plant.

The term “hybridization” as used herein is generally used to meanhybridization of nucleic acids at appropriate conditions of stringencyas would be readily evident to those skilled in the art depending uponthe nature of the probe sequence and target sequences. Conditions ofhybridization and washing are well known in the art, and the adjustmentof conditions depending upon the desired stringency by varyingincubation time, temperature and/or ionic strength of the solution arereadily accomplished. See, for example, Sambrook, J. et al., MolecularCloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Press,Cold Spring Harbor, N.Y., 1989. The choice of conditions is dictated bythe length of the sequences being hybridized, in particular, the lengthof the probe sequence, the relative G-C content of the nucleic acids andthe amount of mismatches to be permitted. Low stringency conditions arepreferred when partial hybridization between strands that have lesserdegrees of complementarity is desired. When perfect or near perfectcomplementarity is desired, high stringency conditions are preferred.For typical high stringency conditions, the hybridization solutioncontains 6× S.S.C., 0.01 M EDTA, 1× Denhardt's solution and 0.5% SDS.Hybridization is carried out at about 68° C. for about 3 to 4 hours forfragments of cloned DNA and for about 12 to about 16 hours for totaleukaryotic DNA. For lower stringencies the temperature of hybridizationis reduced to about 42° C. below the melting temperature (T_(M)) of theduplex. The T_(M) is known to be a function of the G-C content andduplex length as well as the ionic strength of the solution.

As used herein, the term “substantial sequence identity” or “substantialhomology” is used to indicate that a nucleotide sequence or an aminoacid sequence exhibits substantial structural or functional equivalencewith another nucleotide or amino acid sequence. Any structural orfunctional differences between sequences having substantial sequenceidentity or substantial homology will be de minimis; that is, they willnot affect the ability of the sequence to function as indicated in thedesired application. Differences may be due to inherent variations incodon usage among different species, for example. Structural differencesare considered de minimis if there is a significant amount of sequenceoverlap or similarity between two or more different sequences or if thedifferent sequences exhibit similar physical characteristics even if thesequences differ in length or structure. Such characteristics include,for example, ability to hybridize under defined conditions, or in thecase of proteins, immunological crossreactivity, similar enzymaticactivity, etc. Each of these characteristics can readily be determinedby the skilled practitioner by art known methods.

Additionally, two nucleotide sequences are “substantially complementary”if the sequences have at least about 70 percent, more preferably, 80percent and most preferably about 90 percent sequence similarity betweenthem. Two amino acid sequences are substantially homologous if they haveat least 50%, preferably 70% similarity between the active portions ofthe polypeptides.

As used herein, the phrase “hybridizes to a corresponding portion” of aDNA or RNA molecule means that the molecule that hybridizes, e.g.,oligonucleotide, polynucleotide, or any nucleotide sequence (in sense orantisense orientation) recognizes and hybridizes to a sequence inanother nucleic acid molecule that is of approximately the same size andhas enough sequence similarity thereto to effect hybridization underappropriate conditions. For example, a 100 nucleotide long antisensemolecule from the 3′ coding or non-coding region of tomato DHS willrecognize and hybridize to an approximately 100 nucleotide portion of anucleotide sequence within the 3′ coding or non-coding region,respectively of carnation DHS gene or any other plant DHS gene so longas there is about 70% or more sequence similarity between the twosequences. It is to be understood that the size of the “correspondingportion” will allow for some mismatches in hybridization such that the“corresponding portion” may be smaller or larger than the molecule whichhybridizes to it, for example 20-30% larger or smaller, preferably nomore than about 12-15% larger or smaller.

The term “functional derivative” of a nucleic acid (or poly- oroligonucleotide) is used herein to mean a fragment, variant, homolog, oranalog of the gene or nucleotide sequence encoding senescence-inducedDHS or senescence-induced eIF-5A. A functional derivative may retain atleast a portion of the function of the senescence-induced DHS or eIF-5Aencoding DNA which permits its utility in accordance with the invention.Such function may include the ability to hybridize under low stringencyconditions with native tomato, Arabidopsis or carnationsenescence-induced DHS or eIF-5A or substantially homologous DNA fromanother plant which encodes senescence-induced DHS or eIF-5A or with anmRNA transcript thereof, or, in antisense orientation, to inhibit thetranscription and/or translation of plant senescence-induced DHS oreIF-5A mRNA, or the like.

A “fragment” of the gene or DNA sequence refers to any subset of themolecule, e.g., a shorter polynucleotide or oligonucleotide. A “variant”refers to a molecule substantially similar to either the entire gene ora fragment thereof, such as a nucleotide substitution variant having oneor more substituted nucleotides, but which maintains the ability tohybridize with the particular gene or to encode mRNA transcript whichhybridizes with the native DNA. A “homolog” refers to a fragment orvariant sequence from a different plant genus or species. An “analog”refers to a non-natural molecule substantially similar to or functioningin relation to either the entire molecule, a variant or a fragmentthereof.

By “altered expression” or “modified expression” of a gene, e.g., thesenescence-induced DHS gene or senescence-induced eIF-5A gene, is meantany process or result whereby the normal expression of the gene, forexample, that expression occurring in an unmodified fruit bearing,flowering or other plant, is changed in some way. As intended herein,alteration in gene expression is complete or partial reduction in theexpression of the senescence-induced DHS gene or senescence-inducedeIF-5A gene or both, but may also include a change in the timing ofexpression, or another state wherein the expression of thesenescence-induced DHS gene or senescence-induced eIF-5A gene or bothdiffers from that which would be most likely to occur naturally in anunmodified plant or cultivar. A preferred alteration is one whichresults in reduction of senescence-induced DHS production,senescence-induced eIF-5A production or both by the plant compared toproduction in an unmodified plant.

In producing a genetically altered plant in accordance with thisinvention, it is preferred to select individual plantlets or plants bythe desired trait, generally reduced senescence-induced DHS expressionor production or reduced senescence-induced eIF-5A expression or both.Expression of senescence-induced DHS and senescence-induced eIF-5A canbe determined, for example by observations of delayed or reducedsenescence in transgenic plants. It is also possible to quantitate theactivity of DHS and/or eIF-5A in transgenic plants in comparison tocontrol (normal, non-transgenic) plants using known assays.

In order for a newly inserted gene or DNA sequence to be expressed,resulting in production of the protein which it encodes, or in the caseof antisense DNA, to be transcribed, resulting in an antisense RNAmolecule, the proper regulatory elements should be present in properlocation and orientation with respect to the gene or DNA sequence. Theregulatory regions may include a promoter, a 5′-non-translated leadersequence and a 3′-polyadenylation sequence as well as enhancers andother regulatory sequences.

Promoter regulatory elements that are useful in combination with thesenescence-induced DHS gene to generate sense or antisense transcriptsof the gene include any plant promoter in general, and moreparticularly, a constitutive promoter such as the fig wart mosaic virus35S promoter, the cauliflower mosaic virus promoter, CaMV35S promoter,or the MAS promoter, or a tissue-specific or senescence-inducedpromoter, such as the carnation petal GST1 promoter or the ArabidopsisSAG12 promoter (See, for example, J. C. Palaqui et al., Plant Physiol.,112:1447-1456 (1996); Morton et al., Molecular Breeding, 1:123-132(1995); Fobert et al., Plant Journal, 6:567-577 (1994); and Gan et al.,Plant Physiol., 113:313 (1997), incorporated herein by reference).Preferably, the promoter used in the present invention is a constitutivepromoter, most preferably a double 35S promoter is used.

Expression levels from a promoter which is useful for the presentinvention can be tested using conventional expression systems, forexample by measuring levels of a reporter gene product, e.g., protein ormRNA in extracts of the leaves, flowers, fruit or other tissues of atransgenic plant into which the promoter/reporter gene have beenintroduced.

The present invention provides antisense oligonucleotides andpolynucleotides complementary to the gene encoding tomatosenescence-induced DHS, carnation senescence-induced DHS, Arabidopsissenescence-induced DHS or complementary to a gene or gene fragment fromanother plant, which hybridizes with the tomato, carnation orArabidopsis senescence-induced DHS gene under low to high stringencyconditions. The present invention also provides antisenseoligonucleotides and polynucleotides complementary to the gene encodingtomato senescence-induced eIF-5A, carnation senescence-induced eIF-5A,Arabidopsis senescence-induced eIF-5A or complementary to a gene or genefragment from another plant, which hybridizes with the tomato, carnationor Arabidopsis senescence-induced eIF-5A gene under low to highstringency conditions. Such antisense oligonucleotides should be atleast about six nucleotides in length to provide minimal specificity ofhybridization and may be complementary to one strand of DNA or mRNAencoding the senescence-induced gene or a portion thereof, or toflanking sequences in genomic DNA which are involved in regulatingsenescence-induced DHS or eIF-5A gene expression. The antisenseoligonucleotide may be as large as 100 nucleotides or more and mayextend in length up to and beyond the full coding sequence for which itis antisense. The antisense oligonucleotides can be DNA or RNA orchimeric mixtures or derivatives or modified versions thereof, singlestranded or double stranded.

The action of the antisense oligonucleotide may result in alteration,primarily inhibition, of senescence-induced DHS expression,senescence-induced eIF-5A expression or both in cells. For a generaldiscussion of antisense see: Alberts, et al., Molecular Biology of theCell, 2nd ed., Garland Publishing, Inc. New York, N.Y., 1989 (inparticular pages 195-196, incorporated herein by reference).

The antisense oligonucleotide may be complementary to any correspondingportion of the senescence-induced DHS or eIF-5A gene. In one embodiment,the antisense oligonucleotide may be between 6 and 100 nucleotides inlength, and may be complementary to the 5′-non-coding or sequenceswithin the 3′-end of the senescence-induced DHS or eIF-5A sequence, forexample. Antisense oligonucleotides primarily complementary to5′-non-coding sequences are known to be effective inhibitors ofexpression of genes encoding transcription factors. Branch, M. A.,Molec. Cell Biol., 13:4284-4290 (1993).

Preferred antisense oligonucleotides are substantially complementary toa portion of the mRNA encoding senescence-induced DHS orsenescence-induced eIF-5A, the portion of the mRNA being approximatelythe same size as the antisense oligonuleotide. For example, introductionof the full length cDNA clone encoding senescence-induced DHS or eIF-5Ain an antisense orientation into a plant is expected to result insuccessfully altered senescence-induced DHS and/or eIF-5A geneexpression. Moreover, as demonstrated in FIGS. 21-35 introduction ofpartial sequences, targeted to specific portions of thesenescence-induced DHS gene or senescence-induced eIF-5A gene or both,can be equally effective.

The minimal amount of homology required by the present invention is thatsufficient to result in sufficient complementarity to providerecognition of the specific target RNA or DNA and inhibition orreduction of its translation or function while not affecting function ofother RNA or DNA molecules and the expression of other genes. While theantisense oligonucleotides of the invention comprise sequencescomplementary to a corresponding portion of an RNA transcript of thesenescence-induced DHS gene or senescence-induced eIF-5A gene, absolutecomplementarity, although preferred is not required. The ability tohybridize may depend on the length of the antisense oligonucleotide andthe degree of complementarity. Generally, the longer the hybridizingnucleic acid, the more base mismatches with the senescence-induced DHStarget sequence it may contain and still form a stable duplex. Oneskilled in the art can ascertain a tolerable degree of mismatch by useof standard procedures to determine the melting temperature of thehybridized complex, for example.

The antisense RNA oligonucleotides may be generated intracellularly bytranscription from exogenously introduced nucleic acid sequences. Theantisense molecule may be delivered to a cell by transformation ortransfection or infection with a vector, such as a plasmid or virus intowhich is incorporated DNA encoding the antisense senescence-induced DHSsequence operably linked to appropriate regulatory elements, including apromoter. Within the cell the exogenous DNA sequence is expressed,producing an antisense RNA of the senescence-induced DHS gene.

Vectors can be plasmids, preferably, or may be viral or other vectorsknown in the art to replicate and express genes encoded thereon in plantcells or bacterial cells. The vector becomes chromosomally integratedsuch that it can be transcribed to produce the desired antisensesenescence-induced DHS RNA. Such plasmid or viral vectors can beconstructed by recombinant DNA technology methods that are standard inthe art. For example, the vector may be a plasmid vector containing areplication system functional in a prokaryotic host and an antisenseoligonucleotide or polynucleotide according to the invention.Alternatively, the vector may be a plasmid containing a replicationsystem functional in Agrobacterium and an antisense oligonucleotide orpolynucleotide according to the invention. Plasmids that are capable ofreplicating in Agrobacterium are well known in the art. See, Miki, etal., Procedures for Introducing Foreign DNA Into Plants, Methods inPlant Molecular Biology and Biotechnology, Eds. B. R. Glick and J. E.Thompson. CRC Press (1993), PP. 67-83.

The tomato DHS gene was cloned in antisense orientation into a plasmidvector in the following manner. The pCD plasmid, which is constructedfrom a pUC18 backbone and contains the 35S promoter from cauliflowermosaic virus (CaMV) followed by a multiple cloning site and an octapinesynthase termination sequence was used for cloning the tomato DHS gene.The pCd-DHS (antisense) plasmid was constructed by subcloning the fulllength tomato DHS gene in the antisense orientation into the pCD plasmidusing XhoI and SacI restriction sites.

An oligonucleotide, preferably between about 6 and about 100 nucleotidesin length and complementary to the target sequence of senescence-inducedDHS or senescence-induced eIF-5A gene, may be prepared by recombinantnucleotide technologies or may be synthesized from mononucleotides orshorter oligonucleotides, for example. Automated synthesizers areapplicable to chemical synthesis of the oligo- and polynucleotides ofthe invention. Procedures for constructing recombinant nucleotidemolecules in accordance with the present invention are disclosed inSambrook, et al., In: Molecular Cloning: A Laboratory Manual, 2nd ed.,Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), which isincorporated herein in its entirety. Oligonucleotides which encodeantisense RNA complementary to senescence-induced deoxyhypusine synthasesequence can be prepared using procedures well known to those in theart. Details concerning such procedures are provided in Maniatis, T. etal., Molecular mechanisms in the Control of Gene expression, eds.,Nierlich, et al., eds., Acad. Press, N.Y. (1976).

In an alternative embodiment of the invention, inhibition of expressionof endogenous plant senescence-induced DHS, senescence-induced eIF-5A orboth is the result of co-suppression through over-expression of anexogenous senescence-induced DHS or eIF-5A gene or gene fragment or bothintroduced into the plant cell. In this embodiment of the invention, avector encoding senescence-induced DHS, senescence-induced eIF-5A orboth in the sense orientation is introduced into the cells in the samemanner as described herein for antisense molecules. Preferably, thesenescence-induced DHS or senescence-induced eIF-5A is operativelylinked to a strong constitutive promoter, such as for example the figwart mosaic virus promoter or CaMV35S or a double 35 S promoter.

In another embodiment of the invention, inhibition of expression ofendogenous plant senescence-induced DHS, senescence-induced eIF-5A orboth is effected through the use of ribozymes. Ribozymes are RNAmolecules exhibiting sequence-specific endoribonuclease activity. Anexample is the hammerhead ribozyme which cleaves at a UH (where H is anA, C or U residue) recognition site in the target RNA and containsbase-pairing regions that direct the catalytic domain of the ribozyme tothe target site of the substrate RNA. Ribozymes are highlytarget-specific and can be designed to inactivate one member of amultigene family or targeted to conserved regions of related mRNAs. (SeeMerlo et al., The Plant Cell, 10:1603-1621, 1998). The ribozyme moleculemay be delivered to a cell by transformation, transfection or infectionwith a vector, such as a plasmid or virus, into which is incorporatedthe ribozyme operatively linked to appropriate regulatory elements,including a promoter. Such a ribozyme construct contains base-pairingarms that direct it to a cleavage site within the senescence-induced DHSmRNA, or senescence-induced eIF-5A mRNA resulting in cleavage of DHS oreIF-5A mRNA and inhibition of senescence -induced DHS and/or eIF-5Aexpression.

Transgenic plants made in accordance with the present invention may beprepared by DNA transformation using any method of plant transformationknown in the art. Plant transformation methods include directco-cultivation of plants, tissues or cells with Agrobacteriumtumefaciens or direct infection (Miki, et al., Meth. in Plant Mol. Biol.and Biotechnology, (1993), p. 67-88); direct gene transfer intoprotoplasts or protoplast uptake (Paszkowski, et al., EMBO J., 12:2717(1984); electroporation (Fromm, et al., Nature, 319:719 (1986); particlebombardment (Klein et al., BioTechnology, 6:559-563 (1988); injectioninto meristematic tissues of seedlings and plants (De LaPena, et al.,Nature, 325:274-276 (1987); injection into protoplasts of cultured cellsand tissues (Reich, et al., BioTechnology, 4:1001-1004 (1986)).

Generally a complete plant is obtained from the transformation process.Plants are regenerated from protoplasts, callus, tissue parts orexplants, etc. Plant parts obtained from the regenerated plants in whichthe expression of senescence-induced DHS, senescence-induced eIF-5A orboth is altered, such as leaves, flowers, fruit, seeds and the like areincluded in the definition of “plant” as used herein. Progeny, variantsand mutants of the regenerated plants are also included in thedefinition of “plant.”

The tomato, carnation or Arabidopsis senescence-induced DHS protein orfunctional derivatives thereof, and tomato, carnation or Arabidopsissenescence-induced eIF-5A protein or functional derivatives thereof arepreferably produced by recombinant technologies, optionally incombination with chemical synthesis methods. In one embodiment of theinvention the senescence-induced DHS is expressed as a fusion protein,preferably consisting of the senescence-induced DHS fused with maltosebinding protein.

“Functional derivatives” of the senescence-induced DHS orsenescence-induced eIF-5A protein as described herein are fragments,variants, analogs, or chemical derivatives of senescence-induced DHS orsenescence-induced eIF-5A, respectively, which retain at least a portionof the senescence-induced DHS or eIF-5A activity or immunological crossreactivity with an antibody specific for senescence-induced DHS orsenescence-induced eIF-5A, respectively. A fragment of thesenescence-induced DHS or senescence-induced eIF-5A protein refers toany subset of the molecule. Variant peptides may be made by directchemical synthesis, for example, using methods well known in the art. Ananalog of senescence-induced DHS or senescence-induced eIF-5A refers toa non-natural protein substantially similar to either the entire proteinor a fragment thereof. Chemical derivatives of senescence-induced DHS orsenescence-induced-eIF-5A contain additional chemical moieties notnormally a part of the peptide or peptide fragment. Modifications may beintroduced into peptides or fragments thereof by reacting targeted aminoacid residues of the peptide with an organic derivatizing agent that iscapable of reacting with selected side chains or terminal residues.

A senescence-induced DHS or senescence-induced eIF-5A protein or peptideaccording to the invention may be produced by culturing a celltransformed with a nucleotide sequence of this invention (in the senseorientation), allowing the cell to synthesize the protein and thenisolating the protein, either as a free protein or as a fusion protein,depending on the cloning protocol used, from either the culture mediumor from cell extracts. Alternatively, the protein can be produced in acell-free system. Ranu, et al., Meth. Enzymol., 60:459-484, (1979).

Having now generally described the invention, the same will be morereadily understood through reference to the following examples which areprovided by way of illustration, and are not intended to be limiting tothe present invention.

EXAMPLE 1

Messenger RNA (mRNA) Isolation

Total RNA was isolated from tomato flowers and tomato fruit at variousdevelopmental stages and from leaves (untreated or after chilling orsorbitol treatment). Briefly, the tissue (5 g) was ground in liquidnitrogen. The ground powder was mixed with 30 ml guanidinium buffer (4 Mguanidinium isothiocyanate, 2.5 mM NaOAc pH 8.5, 0.8%β-mercaptoethanol). The mixture was filtered through four layers ofcheesecloth and centrifuged at 10,000×g at 4° C. for 30 minutes. Thesupernatant was then subjected to cesium chloride density gradientcentrifugation at 26,000×g for 20 hours. The pelleted RNA was rinsedwith 75% ethanol, resuspended in 600 μl DEPC-treated water and the RNAprecipitated at −70° C. with 0.75 ml 95% ethanol and 30 μl of 3M NaOAc.Ten μg of RNA were fractionated on a 1.2% denaturing formaldehydeagarose gel and transferred to a nylon membrane. Randomly primed³²P-dCTP-labelled full length DHS cDNA (SEQ ID NO:1) was used to probethe membrane at 42° C. overnight. The membrane was then washed once in1×SSC containing 0.1% SDS at room temperature for 15 minutes and threetimes in 0.2×SSC containing 0.1% SDS at 65° C. for 15 minutes each. Themembrane was exposed to x-ray film overnight at −70° C.

PolyA⁺ mRNA was isolated from total RNA using the PolyA⁺ tract mRNAIsolation System available from Promega. PolyA⁺ mRNA was used as atemplate for cDNA synthesis using the ZAP Express® cDNA synthesis systemavailable from Stratagene (La Jolla, Calif.)

Tomato Leaf cDNA Library Screening

A cDNA library made using mRNA isolated from Match F1 hybrid tomatoleaves that had been exposed to 2 M sorbitol for six hours was dilutedto approximately 5×10⁶ PFU/ml. The cDNA library was screened using a³²P-labelled 600 bp RT-PCR fragment. Three positive cDNA clones wereexcised and recircularized into a pBK-CMV® (Stratagene) phagemid usingthe method in the manufacturer's instructions. The full length cDNA wasinserted into the pBK-CMV vector.

Plasmid DNA Isolation, DNA Sequencing

The alkaline lysis method described by Sambrook et al., (Supra) was usedto isolate plasmid DNA. The full length positive cDNA clone wassequenced using the dideoxy sequencing method. Sanger, et al., Proc.Natl. Acad. Sci. USA, 74:5463-5467. The open reading frame was compiledand analyzed using BLAST search (GenBank, Bethesda, Md.) and alignmentof the five most homologous proteins with the derived amino acidsequence of the encoded gene was achieved using a BCM Search Launcher:Multiple Sequence Alignments Pattern-Induced Multiple Alignment Method(See F. Corpet, Nuc. Acids Res., 16:10881-10890, (1987)). Functionalmotifs present in the derived amino acid sequence were identified byMultiFinder.

Northern Blot Hybridizations of Tomato RNA

Ten μg of total RNA isolated from tomato flowers at various stages (budand blossom and senescing petals that are open widely or drying), tomatoleaves, and tomato fruit at various stages of ripening (breaker, i.e.,green fruit with less than 10% red color, pink, i.e., the entire fruitis orange or pink, and red, either soft or firm) were separated on 1%denatured formaldehyde agarose gels and immobilized on nylon membranes.The full length tomato cDNA labelled with ³²P-dCTP using a random primerkit (Boehringer Mannheim) was used to probe the filters (7×10⁷ cpm). Thefilters were washed once with 1×SSC, 0.1% SDS at room temperature andthree times with 0.2×SSC, 0.1% SDS at 65° C. The filters were dried andexposed to X-ray film overnight at −70° C. The results are shown inFIGS. 6, 7, 8 and 9.

Northern Blot Hybridization of Arabidopsis RNA

Total RNA from leaves of Arabidopsis plants at five weeks of age (lane1), six weeks (lane 2) and seven weeks (lane 3) was isolated as above,separated on 1% denatured formaldehyde agarose gels and immobilized onnylon membranes. The full-length Arabidopsis senescence-induced DHS cDNAlabelled with ³²P-dCTP using a random primer kit (Boehringer Mannheim)was used to probe the filters (7×10⁷ cpm). The filters were washed oncewith 1×SSC, 0.1% SDS at room temperature and three times with 0.2×SSC,0.1% SDS at 65° C. The filters were dried and exposed to X-ray filmovernight at −70° C. The results are shown in FIG. 11.

Northern Blot Hybridization of Carnation RNA

Total RNA from petals of carnation plants at various stages of flowerdevelopment, i.e., tight-bud flowers (lane 1), beginning to open (lane2), fully open flowers (lane 3), flowers with inrolling petals (lane 4),was isolated as above, separated on 1% denatured formaldehyde agarosegels and immobilized on nylon membranes. The full-length carnationsenescence-induced DHS cDNA labelled with ³²P-dCTP using a random primerkit (Boehringer Mannheim) was used to probe the filters (7×10⁷ cpm). Thefilters were washed once with 1×SSC, 0.1% SDS at room temperature andthree times with 0.2×SSC, 0.1% SDS at 65° C. The filters were dried andexposed to X-ray film overnight at −70° C. The results are shown in FIG.12.

EXAMPLE 2

Sorbitol Induction of Tomato Senescence-Induced DHS Gene

Tomato leaves were treated with 2 M sorbitol in a sealed chamber for sixhours. RNA was extracted from the sorbitol treated leaves as follows.

Leaves (5 g) were ground in liquid nitrogen. The ground powder was mixedwith 30 ml guanidinium buffer (4 M guanidinium isothiocyanate, 2.5 mMNaOAc pH 8.5, 0.8% β-mercaptoethanol). The mixture was filtered throughfour layers of cheesecloth and centrifuged at 10,000×g at 4° C. for 30minutes. The supernatant was then subjected to cesium chloride densitygradient centrifugation at 26,000×g for 20 hours. The pelleted RNA wasrinsed with 75% ethanol, resuspended in 600 μl DEPC-treated water andthe RNA precipitated at −70° C. with 0.75 ml 95% ethanol and 30 μl of 3MNaOAc. Ten μg of RNA were fractionated on a 1.2% denaturing formaldehydeagarose gel and transferred to a nylon membrane. Randomly primed³²P-dCTP-labelled full length DHS cDNA (SEQ ID NO:1) was used to probethe membrane at 42° C. overnight. The membrane was then washed once in1×SSC containing 0.1% SDS at room temperature for 15 minutes and threetimes in 0.2×SSC containing 0.1% SDS at 65° C. for 15 minutes each. Themembrane was exposed to x-ray film overnight at −70° C.

The results are shown in FIG. 8. As can be seen, transcription of DHS isinduced in leaves by sorbitol.

EXAMPLE 3

Induction of the Tomato DHS Gene in Senescing Flowers

Tight flower buds and open, senescing flowers of tomato plants wereharvested, and RNA was isolated as in Example 2. Ten μg RNA werefractionated on a 1.2% denaturing formaldehyde agarose gel andtransferred to a nylon membrane. Randomly primed ³²P-dCTP-labelled fulllength DHS cDNA (SEQ ID NO.1) was used to probe the membrane at 42° C.overnight. The membrane then was washed once in 1×SSC containing 0.1%SDS at room temperature for 15 minutes and then washed three times in0.2×SSC containing 0.1% SDS at 65° C. for fifteen minutes each. Themembrane was exposed to x-ray film overnight at −70° C.

The results are shown in FIG. 6. As can be seen, transcription of DHS isinduced in senescing flowers.

EXAMPLE 4

Induction of the Tomato DHS Gene in Ripening Fruit

RNA was isolated from breaker, pink and ripe fruit as in Example 2. Tenμg RNA were fractionated on a 1.2% denaturing formaldehyde agarose geland transferred to a nylon membrane. Randomly primed ³²P-dCTP-labelledfull length DHS cDNA (SEQ ID NO.1) (FIG. 1) was used to probe themembrane at 42° C. overnight. The membrane then was washed once in 1×SSCcontaining 0.1% SDS at room temperature for 15 minutes and then washedthree times in 0.2×SSC containing 0.1% SDS at 65° C. for fifteen minuteseach. The membrane was exposed to x-ray film overnight at −70° C.

The results are shown in FIG. 7. As can be seen, transcription of DHS isstrongest in ripe, red fruit just prior to the onset of senescenceleading to spoilage.

EXAMPLE 5

Induction of Tomato Senescence-Induced DHS Gene by Chilling

Tomato plants in pots (7-8 weeks old) were exposed to 6° C. for twodays, three days or six days in a growth chamber. The light cycle wasset for eight hours of dark and sixteen hours of light. Plants wererewarmed by moving them back into a greenhouse. Plants that were notrewarmed were harvested immediately after removal from the growthchamber. RNA was extracted from the leaves as follows.

Leaves (5 g) were ground in liquid nitrogen. The ground powder was mixedwith 30 ml guanidinium buffer (4 M guanidinium isothiocyanate, 2.5 mMNaOAc pH 8.5, 0.8% β-mercaptoethanol). The mixture was filtered throughfour layers of cheesecloth and centrifuged at 10,000 g at 4° C. for 30minutes. The supernatant was then subjected to cesium chloride densitygradient centrifugation at 26,000 g for 20 hours. The pelleted RNA wasrinsed with 75% ethanol, resuspended in 600 μl DEPC-treated water andthe RNA precipitated at −70° C. with 0.75 ml 95% ethanol and 30 μl of 3MNaOAc. Ten μg of RNA were fractionated on a 1.2% denaturing formaldehydeagarose gel and transferred to a nylon membrane. Randomly primed³²P-dCTP-labelled full length DHS cDNA (SEQ ID NO:1) was used to probethe membrane at 42° C. overnight. The membrane was then washed once in1×SSC containing 0.1% SDS at room temperature for 15 minutes and threetimes in 0.2×SSC containing 0.1% SDS at 65° C. for 15 minutes each. Themembrane was exposed to x-ray film overnight at −70° C.

The results are shown in FIG. 9. As can be seen, transcription of DHS isinduced in leaves by exposure to chilling temperature and subsequentrewarming, and the enhanced transcription correlates with chillingdamage measured as membrane leakiness.

EXAMPLE 6

Generation of an Arabidopsis PCR Product Using Primers Based onUnidentified

Arabidopsis Genomic Sequence

A partial length senescence-induced DHS sequence from an ArabidopsiscDNA template was generated by PCR using a pair of oligonucleotideprimers designed from Arabidopsis genomic sequence. The 5′ primer is a19-mer having the sequence, 5′-GGTGGTGTTGAGGAAGATC (SEQ ID NO:7); the 3′primer is a 20 mer having the sequence, GGTGCACGCCCTGATGAAGC-3′ (SEQ IDNO:8). A polymerase chain reaction using the Expand High Fidelity PCRSystem (Boehringer Mannheim) and an Arabidopsis senescing leaf cDNAlibrary as template was carried out as follows.

Reaction components: cDNA 1 μl (5 × 10⁷ pfu) dNTP (10 mM each) 1 μlMgCl₂ (5 mM) + 10x buffer 5 μl Primers 1 and 2 (100 μM each) 2 μl ExpandHigh Fidelity DNA polymerase 1.75 U Reaction volume 50 μl Reactionparamaters: 94° C. for 3 min 94° C./1 min, 58° C./1 min, 72° C./2 min,for 45 cycles 72° C. for 15 min.

EXAMPLE 7

Isolation of Genomic DNA and Southern Analysis

Genomic DNA was extracted from tomato leaves by grinding 10 grams oftomato leaf tissue to a fine powder in liquid nitrogen. 37.5 ml of amixture containing 25 ml homogenization buffer [100 mM Tris-HCl, pH 8.0,100 mm EDTA, 250 mM NaCl, 1% sarkosyl, 1% 2-mercaptoethanol, 10 μg/mlRNase and 12.5 ml phenol] prewarmed to 60° C. was added to the groundtissue. The mixture was shaken for fifteen minutes. An additional 12.5ml of chloroform/isoamyl alcohol (24:1) was added to the mixture andshaken for another 15 minutes. The mixture was centrifuged and theaqueous phase reextracted with 25 ml phenol/chloroform/isoamylalcohol(25:24:1) and chloroform/isoamylalcohol (24:1). The nucleic acids wererecovered by precipitaion with 15 ml isopropanol at room temperature.The precipitate was resuspended in 1 ml of water.

Genomic DNA was subjected to restriction enzyme digestion as follows: 10μg genomic DNA, 40 μl 10× reaction buffer and 100 U restriction enzyme(XbaI, EcoRI, EcoRV or HinDIII) were reacted for five to six hours in atotal reaction volume of 400 μl. The mixture was then phenol-extractedand ethanol-precipitated. The digested DNA was subjected to agarose gelelectrophoresis on a 0.8% agarose gel at 15 volts for approximately 15hours. The gel was submerged in denaturation buffer [87.66 g NaCl and 20g NaOH/Liter] for 30 minutes with gentle agitation, rinsed in distilledwater and submerged in neutralization buffer [87.66 g NaCl and 60.55 gtris-HCl, pH 7.5/Liter] for 30 minutes with gentle agitation. The DNAwas transferred to a Hybond-N⁺ nylon membrane by capillary blotting.

Hybridization was performed overnight at 42° C. using 1×10⁶ cpm/ml of³²P-dCTP-labeled full length DHS cDNA or 3′-non-coding region of the DHScDNA clone. Prehybridization and hybridization were carried out inbuffer containing 50% formamide, 6×SSC, 5× Denhardt's solution, 0.1% SDSand 100 mg/ml denatured salmon sperm DNA. The membrane was prehybridizedfor two to four hours; hybridization was carried out overnight.

After hybridization was complete, membranes were rinsed at roomtemperature in 2×SSC and 0.1% SDS and then washed in 2×SSC and 0.1% SDSfor 15 minutes and 0.2×SSC and 0.1% SDS for 15 minutes. The membrane wasthen exposed to x-ray film at −80° C. overnight. The results are shownin FIG. 5.

EXAMPLE 8

Isolation of a Senescence-Induced eIF-5A Gene from Arabidopsis

A full-length cDNA clone of the senescence-induced eIF-5A gene expressedin Arabidopsis leaves was obtained by PCR using an Arabidopsis senescingleaf cDNA library as template. Initially, PCR products corresponding tothe 5′- and 3′-ends of the gene were made using a degenerate upstreamprimer <AAARRYCGMCCYTGCAAGGT>(SEQ ID NO:17) paired with vector T7 primer<AATACGACTCACTATAG> (SEQ ID NO:18), and a degenerate downstream primer<TCYTTNCCYTCMKCTAAHCC> (SEQ ID NO:19) paired with vector T3 primer<ATTAACCCTCACTAAAG> (SEQ ID NO: 20). The PCR products were subclonedinto pBluescript for sequencing. The full-length cDNA was then obtainedusing a 5′-specific primer <CTGTTACCAAAAAATCTGTACC> (SEQ ID NO: 21)paired with a 3′-specific primer <AGAAGAAGTATAAAAACCATC> (SEQ ID NO:22), and subcloned into pBluescript for sequencing.

EXAMPLE 9

Isolation of a Senescence-Induced eIF-5A Gene from Tomato Fruit

A full-length cDNA clone of the senescence-induced eIF-5A gene expressedin tomato fruit was obtained by PCR using a tomato fruit cDNA library astemplate. Initially, PCR products corresponding to the 5′- and 3′-endsof the gene were made using a degenerate upstream primer (SEQ ID NO:17)paired with vector T7 primer (SEQ ID NO:18), and a degenerate downstreamprimer (SEQ ID NO:19) paired with vector T3 primer (SEQ ID NO: 20). ThePCR products were subcloned into pBluescript for sequencing. Thefull-length cDNA was then obtained using a 5′-specific primer<AAAGAATCCTAGAGAGAGAAAGG> (SEQ ID NO: 23) paired with vector T7 primer(SEQ ID NO: 18), and subcloned into pBluescript for sequencing.

EXAMPLE 10

Isolation of a Senescence-Induced eIF-5A Gene from Carnation

A full-length cDNA clone of the senescence-induced eIF-5A gene expressedin carnation flowers was obtained by PCR using a carnation senescingflower cDNA library as template. Initially, PCR products correspondingto the 5′- and 3′-ends of the gene were made using a degenerate upstreamprimer (SEQ ID NO:17) paired with vector T7 primer (SEQ ID NO:18), and adegenerate downstream primer (SEQ ID NO:19) paired with vector T3 primer(SEQ ID NO: 20). The PCR products were subcloned into pBluescript forsequencing. The full-length cDNA was then obtained using a 5′-specificprimer <TTTTACATCAATCGAAAA> (SEQ ID NO: 24) paired with a 3′-specificprimer <ACCAAAACCTGTGTTATAACTCC> (SEQ ID NO: 25), and subcloned intopBluescript for sequencing.

EXAMPLE 11

Isolation of a Senescence-Induced DHS Gene from Arabidopsis

A full-length cDNA clone of the senescence-induced DHS gene expressed inArabidopsis leaves was obtained by screening an Arabidopsis senescingleaf cDNA library. The sequence of the probe (SEQ ID NO: 26) that wasused for screening is shown in FIG. 38. The probe was obtained by PCRusing the senescence leaf cDNA library as a template and primers(indicated as underlined regions in FIG. 38) designed from theunidentified genomic sequence (AB017060) in GenBank. The PCR product wassubcloned into pBluescript for sequencing.

EXAMPLE 12

Isolation of a Senescence-Induced DHS Gene from Carnation

A full-length cDNA clone of the senescence-induced DHS gene expressed incarnation petals was obtained by screening a carnation senescing petalcDNA library. The sequence of the probe (SEQ ID NO: 27) that was usedfor screening is shown in FIG. 39. The probe was obtained by PCR usingthe senescence petal cDNA library as a template and degenerate primers(upstream: 5′ TTG ARG AAG ATY CAT MAA RTG CCT 3′) (SEQ ID NO: 28);downstream: 5′ CCA TCA AAY TCY TGK GCR GTG TT 3′) (SEQ ID NO: 29)). ThePCR product was subcloned into pBluescript for sequencing.

EXAMPLE 13

Transformation of Arabidopsis with Full-Length or 3′ Region ofArabidopsis DHS in

Antisense Orientation

Agrobacteria were transformed with the binary vector, pKYLX71,containing the full-length senescence-induced Arabidopsis DHS cDNAsequence or the 3′ end of the DHS gene (SEQ ID NO:30) (FIG. 36), bothexpressed in the antisense configuration, under the regulation of double35S promoter. Arabidopsis plants were transformed with the transformedAgrobacteria by vacuum infiltration, and transformed seeds fromresultant T₀ plants were selected on ampicillin.

FIGS. 21 through 24 are photographs of the transformed Arabidopsisplants, showing that expression of the DHS gene or 3′ end thereof inantisense orientation in the transformed plants results in increasedbiomass, e.g., larger leaves and increased plant size. FIG. 25illustrates that the transgenic Arabidopsis plants have increased seedyield.

EXAMPLE 14

Transformation of Tomato Plants with Full-Length or 3′ Region of TomatoDHS in

Antisense Orientation

Agrobacteria were transformed with the binary vector, pKYLX71,containing the full-length senescence-induced tomato DHS cDNA sequenceor the 3′ end of the DHS gene (SEQ ID NO:31) (FIG. 37), both expressedin the antisense configuration, under the regulation of double 35Spromoter. Tomato leaf explants were formed with these Agrobacteria, andtransformed callus and plantlets were generated and selected by standardtissue culture methods. Transformed plantlets were grown to maturefruit-producing T₁ plants under greenhouse conditions.

FIGS. 26 through 35 are photographs showing that reduced expression ofthe senescence-induced tomato DHS gene in the transformed plants resultsin increased biomass, e.g., larger leaf size and larger plants as seenin the transformed Arabidopsis plants, as well as delayed softening andspoilage of tomato fruit.

1. An isolated antisense polynucleotide of SEQ ID NO:5 whereinpolynucleotide inhibits expression of an endogenous senescence-induceddeoxyhypusine synthase gene comprising SEQ ID NO:5 when said antisensepolynucleotide is transcribed in a plant cell.
 2. A vector fortransformation of plant cells comprising (a) the antisensepolynucleotide of claim 1; and (b) regulatory sequences operativelylinked to the antisense polynucleotide such that the antisensepolynucleotide is transcribe in a plant cell into which it istransformed.
 3. The vector according to claim 2 wherein the regulatorysequences comprise a promoter and a transcription termination region. 4.The vector according to claim 2 wherein the regulatory sequencescomprise a constitutive promoter.
 5. The vector according to claim 2wherein the regulatory sequences comprise a plant tissue-specificpromoter.
 6. The vector according to claim 2 wherein the regulatorysequences comprise a senescence-induced plant promoter.
 7. The vectoraccording to claim 2 wherein the regulatory sequences comprise a viralpromoter.
 8. The antisense polynucleotide according to claim 1 whereinthe antisense polynucleotide is the complement of SEQ ID NO:30.
 9. Abacterial cell transformed with the vector according to claim
 2. 10. Atransgenic plant cell transformed with the vector according to claim 2.11. A transgenic plant grown from the plant cell of claim
 10. 12. Aplant part obtained from the plant of claim 11 or progeny of said plant,wherein the plant part or progeny comprises the vector of claim 2wherein the plant part is a fruit, flower, or leaf.
 13. A method forinhibiting the expression of endogenous senescence-induced deoxyhypusinesynthase gene comprising SEQ ID NO:5 in a plant, said method comprising(1) integrating into the genome of at least one cell of the plant thevector of claim 2 and (2) growing said plant, whereby said antisensepolynucleotide inhibits expression of the senescence-induceddeoxyhypusine synthase gene comprising SEQ ID NO:5.
 14. The methodaccording to claim 13 wherein said inhibition of expression results inincreased biomass of said plant as compared to biomass of a wild typeplant not having been transformed with the vector of claim
 2. 15. Themethod according to claim 13 wherein said inhibition of expressionresults in delayed fruit softening and spoilage in said plant ascompared to a fruit softening and spoilage of wild type plant not havingbeen transformed with the vector of claim
 2. 16. The method according toclaim 13 wherein said inhibition of expression results in increased seedvolume from said plant as compared to seed volume from a wild type nothaving been transformed with the vector of claim
 2. 17. The methodaccording to claim 13 wherein the regulatory sequences comprise aconstitutive promoter active in the plant.
 18. The method according toclaim 13 wherein the regulatory sequences comprise a tissue specificpromoter active in the plant.
 19. The method according to claim 13wherein the regulatory sequences comprise a senescence-induced promoteractive in the plant.
 20. The method according to claim 13 wherein saidplant is selected from the group consisting of fruit bearing plants andflowering plants.
 21. The method according to claim 13 wherein the plantis a flowering plant.
 22. A plasmid comprising a replication systemfunctional in a prokaryotic host and the antisense polynucleotideaccording to claim
 1. 23. A plasmid comprising a replication systemfunctional in Agrobacterium and the antisense polynucleotide accordingto claim
 1. 24. A vector for transformation of plant cells comprising(a) the antisense polynucleotide of claim 8; and (b) regulatorysequences operatively linked to the antisense polynucleotide such thatthe antisense polynucleotide is transcribed in a plant cell into whichit is transformed.
 25. A method for inhibiting the expression ofendogenous senescence-induced deoxyhypusine synthase gene comprising SEQID NO:5 in a plant, said method comprising (1) integrating into thegenome of at least one cell of the plant the vector of claim 24; and (2)growing said plant, whereby said antisense polynucleotide sequence istranscribed and whereby said transcription of the antisensepolynucleotide inhibits expression of the senescence-induceddeoxyhypusine synthase gene comprising SEQ ID NO:5.